Alcohol-induced liver injury activates hepatic stellate cells (HSCs) resulting in hepatic fibrosis. Withdrawal from alcohol leads to regression of liver fibrosis and disappearance of HSCs/myofibroblasts. We have recently demonstrated that some activated HSCs apoptose, while other HSC inactivate and revert to a quiescent-like phenotype. The overall goals of this Project are to identify the molecular factors that may prevent HSC activation into myofibroblasts, or revert activated HSCs (aHSCs) into a quiescent-like state. Our central hypothesis is that genome wide epigenetic changes regulate HSC phenotype by activation (or suppression) of transcriptional activity in HSCs. We also hypothesize that activation of PPAR?-target genes regulates the quiescent (gHSC) and inactivated (iHSC) phenotypes. AIM1A: We undertake two approaches to improve the model of intragastric ethanol infusion: optimization of the regiment of ethanol and high fat diet (HFD) administration, and utilization of humanized transgenic mice expressing human CYP2E1 protein in Cyp2e1-null mice that make them more susceptible to alcohol-induced liver injury. We hypothesize that these mice also exhibit a defect in HSC inactivation during recovery from alcohol-induced liver fibrosis. AIM1B: We will use a translafional approach to explore whether our findings in mice apply to patients. Using flow cytometry, we will examine if HSC inactivation also occurs in patients. Inactivation of HSCs will be simulated in vitro by TGF-?1 cessation, or in vivo by intrahepatic transplantation of human HSCs into Rag2-/- yc-/- mice. iHSCs will be identified by Vitamin A+?SMAGFAPlowPPAR?hi phenotype. AIM2A: We will use Chip- Seq analysis to access the genome wide methylation and acetylation sites in qHSCs, aHSCs and iHSCs to identify motifs and transcription factors critical for HSC inactivation. Using primary HSC cultures, we will determine if siRNA knockdown or over-expression of these factors blocks HSC activation, or trigger HSC inactivation. AIM2B: We have demonstrated that PPARy is re-expressed in HSCs during inactivafion. To gain a greater insight into mechanisms of HSC inactivation, we will conduct a broad investigation of the epigenetic changes that regulate PPARy-target genes in distinct HSC phenotypes (qHSCs, aHSCs and IHSCs). We will test if functional inhibition of these genes compromises the qHSC and iHSC phenotypes. We anticipate that the results obtained in this study will identify specific factors that can revert aHSC into iHSCs and will give greater insight into mechanisms underlying HSC inactivation, validating potential therapeutic targets that can induce inactivation of already existing HSCs/myofibroblasts in fibrotic liver.